Bioethanol, as a renewable, economically affordable, and environmentally safe energy material, will gradually become a substitute for fossil fuels. It has far-reaching research significance and application value for the development of a sustainable energy strategy (Karimi et al., 2021; Zeng et al., 2021; Ziaei-Rad et al., 2021; Suhartini et al., 2022). Due to competition with food supply in the first generation of bioethanol production, lignocellulose, a non-starch material, has become an important raw material for bioethanol production (Alonso et al., 2019; Maia et al., 2020; Winarni et al., 2020). Adsorption of cellulases onto lignin has been considered as the major factor in retarding enzymatic cellulose degradation of lignocellulosic biomass (Djajadi et al., 2018). Hydrophobic interaction, electrostatic interaction and hydrogen bonding have been regarded as the cause of the nonproductive binding of cellulases to lignin (Djajadi et al., 2018; Li et al., 2020; Song et al., 2020). A natural “biodegradable barrier” of lignin cell walls which are connected in a strong, yet resilient network under the action of covalent and non-covalent bonds render the cellulose inaccessible (Mnich et al., 2020; Chu et al., 2021). Therefore, to reduce the recalcitrance of lignocellulosic biomass to biochemical degradation, pretreatment methods have been developed to break down the lignin-hemicellulose-cellulose matrix and increase the enzyme accessibility of the cellulose scaffold (Jiang et al., 2017a; Jia et al., 2018; Rocha-Martin et al., 2018).

In general, lignin-derived inhibition is the major physical obstacle restricting the enzymatic hydrolysis of cell wall polysaccharides (Leonidas et al., 2019; Zheng et al., 2021). More importantly, the non-specific binding of free cellulase on lignocellulosic substrates may account for the low rate of hydrolysis at the action mechanism level during enzymatic hydrolysis. Some enzymes remain free after the enzymatic hydrolysis of lignocellulosic substrates, while non-specific binding to the residual substrates also prevents the efficient recycling of cellulase (Rahikainen et al., 2011; Kellock et al., 2017; Bhawna et al., 2020). Moreover, the utility of cellulases has been limited due to their low operational stability, high costs, and poor reutilization when used in the native form (Yang et al., 2017).

To overcome these barriers, immobilization is usually used to improve enzyme stability and even activity or selectivity when properly designed, which can also facilitate the reuse of enzymes and effective cost of catalytic processes (Mita and Eldin, 2014; Li et al., 2016; Mehta et al., 2016; Xu et al., 2016; Zhang et al., 2016). The characteristics of various immobilization methods of enzymes is summarized in Table 1. Cellulases represent a large group of enzymes from various organism and with different substrate specificity, biophysical properties, etc. The immobilization behavior is different depending on the enzyme or enzyme mixture investigated. During the immobilization process of cellulase, the structure and properties of carrier materials have significant effects on the performance of the immobilized enzyme (Kalantari et al., 2013; Li et al., 2018). The size of the carriers plays an important role in determining the activity of the immobilized enzyme owing to the inverse relationship between the carrier size and enzyme loading. Thus, large carrier size decreases enzyme activity in general (Valencia et al., 2010), and a reduction in the size of the carriers results in a higher surface area for enzyme binding (Malar et al., 2018; Malar et al., 2020). For the immobilization of cellulase, the smaller size of the surface pore should be kept lower than that of the cellulase macromolecule (6–20 nm), which can further reduce the internal and external diffusion resistance in the heterogeneous system (DiCosimo et al., 2013; Santos et al., 2015). Therefore, nanocarriers are widely used in the immobilization of enzymes because of their unique properties, such as large specific surface area to volume ratio (Cao et al., 2016; Roth et al., 2016; Malar et al., 2020).

Moreover, the immobilization of cellulase has been achieved based on physical adsorption, covalent binding, or affinity interactions (Zang et al., 2014; Hosseini et al., 2018; Zhang and Hay, 2019), including carrier-binding, microemulsion-based organo-gels (MBGs), ultrasonic encapsulation, crosslinking, entrapment, glutathione-labeling, and chelation (Mroczkiewicz et al., 2012; Nicoletti et al., 2015). However, enzymes often display drastically lower activity in organic solvents than in water, and the water layer on the molecular surface of enzymes determines their activity in organic media (Zhang et al., 2012). Therefore, among several approaches to resolve the challenges, one of the most effective methods is immobilization of the enzymes within an aqueous microenvironment in the organic solvents. Microemulsions formed by amphiphilic surfactants have been widely reviewed as effective media for the immobilization of enzymes in hydrophobic solvents (Itabaiana et al., 2014; Rajnish et al., 2021; Savic et al., 2021). The MBGs method based on microemulsions has been used to form matrices for enzyme immobilization to achieve enzymatic catalysis in nonconventional medium as they appear to be rigid and stable for a long time, even within the reaction solution (Zhang et al., 2012). Therefore, the MBGs method has unique advantages of improving the chemical stability of immobilized enzymes and maintaining high catalytic activity (Pavlidis et al., 2010; Itabaiana et al., 2014). It is clearly that the surfactants play an important role in the preparation of nanomaterials (Lou et al., 2017; Bao et al., 2019; Ortiz-Martínez et al., 2019; Alexander et al., 2020).

The surfactants have been widely used for the preparation of nanocarriers as shown in Table 2, forming the nano-template by micelles and emulsions of surfactants is a common method that can greatly reduce the surface tension of the solvent and change the interface composition and structure (Carter and Puig-Sellart, 2016; Bao et al., 2019). Desirable nanostructured materials can be produced because of the special nanoreactors formed by surfactant micelles and the oriented alignment characteristics of surfactants in solution, such as the Langmuir-Blodgett (LB) membranes and liposomes (Lok Kumar et al., 2014; Gutierrez et al., 2016). Furthermore, the non-ionic surfactants can significantly enhance cellulose hydrolysis, thus reducing enzyme loading (Lou et al., 2017; Bao et al., 2019). However, inhibitory effects have been observed with the addition of amphoteric, anionic, and cationic surfactants (Lou et al., 2017; Bao et al., 2019). Moreover, the loss of enzyme activity during immobilization is a notable problem; the structural distortion caused by the strong enzyme-support interactions may produce steric hindrances and catalytic cleft blockage (Carlsson et al., 2014; Suárez et al., 2018). Although a large dose of original cellulase is added for a higher load of immobilized enzyme to improve the activities of the immobilized enzyme, no significant improvement in enzymatic activity has been observed due to the random and inhomogeneous combination of the nanocarriers and cellulase molecules (Nakayama et al., 2009). Oriented immobilization, as a specific binding method, can effectively prevent the nonproductive combination of enzymes and nanocarriers, which further improves the immobilization and hydrolysis efficiency. The reversed micelles formed by surfactants have been successfully used in the preparation of oriented-immobilized lipase when their concentration exceeds the critical micelle concentration (CMC) (Fan et al., 2016). To date, few studies have reported the oriented immobilization of cellulase. Therefore, this review mainly focuses on the important roles of surfactants in the immobilization of cellulase, mainly including the preparation of nanocarriers and cellulase hydrolysis. Moreover, a novel insight into the oriented immobilization of cellulase in a surfactant reversed micelle (SRM) system was discussed and found to have promising prospects.

The structure and properties of carrier materials have great influence on the properties of immobilized cellulase, such as internal geometry (e.g., flat surfaces or thin fibers), specific surface area, superficial activation degree, mechanical resistance, and pore diameter (Santos et al., 2015; Begum et al., 2019; Malar et al., 2020). Meanwhile, partitioning and mass transport limitations may yield spatial variations in local reaction rates in porous materials (Neira and Herr, 2017). Therefore, to improve the stability and catalytic activity of immobilized cellulase, various materials, such as chitin, chitosan, nylon, and polyvinyl alcohol, have been widely used as carriers (Cherian et al., 2015; Priydarshani et al., 2018).

The physical effects of nanocarriers on immobilized cellulase are as follows: 1) The pore size and effective surface area of the nanocarriers. Not all porous carriers can be used for immobilization of cellulase due to the limitation of pore size, which should be larger than or equal to that of the cellulase to reduce steric hindrance. The effective surface area occupied by the enzyme determines the maximum load of the immobilized cellulase (Santos et al., 2015). When a stable surface area is maintained, the amount of immobilized or absorbed cellulases is related to the pore size because the pore diameter determines the size of the protein that can be immobilized on that carrier (Teresa et al., 2009; Webster et al., 2015); 2) the number of carrier-bound active groups (CAGs) is another key factor controlling the enzyme-carrier multi-interaction (Cristina et al., 2011; Santos et al., 2015); 3) the size of carriers plays a very important role in the preparation of immobilized cellulase, in that a smaller carrier size with larger specific surface area will be better for the cellulase immobilization load, and the higher surface porosity of the carriers providing numerous binding sites for cellulase is one of the most important factors influencing the activity of immobilized cellulase (Chen et al., 2010; Santos et al., 2015; Malar et al., 2020); 4) the mechanical properties of the carriers need to be controlled considering the final configuration of the reactor. If the reactor is a fixed-bed reactor, such as inorganic supports like porous glass, silicates, it should possess very high rigidity to withstand high pressures without pressure problems, but the situation is different if a stirred-tank reactor is used (Cristina et al., 2011; Santos et al., 2015); 5) after the cellulase penetrates the carriers, the internal morphology of carriers will determine the possibility of obtaining a very intense or very limited enzyme-carrier interaction (Santos et al., 2015). When the diameter of the carriers is smaller than that of the enzyme, it is difficult to obtain an intense enzyme-carrier interaction (Cristina et al., 2011), but if the carriers have sufficiently large internal surfaces, it is possible to get an intense interaction with a similar flat surface (e.g., agarose beads, porous glass, or silicates) (Malar et al., 2018).

In particular, the special superparamagnetism of magnetic nanocarriers has attracted increasing interest as they allow easy recycling and separation of catalysts and biomolecules from high-viscosity liqueurs and high-solid-content broths. This unique characteristic has been well-applied to immobilization of cellulase, and a better hydrolysis efficiency and recycling feasibility have been observed (Alftrén et al., 2014; Cao et al., 2016; Cipolatti et al., 2014; Xing et al., 2015). During immobilization of cellulase, magnetic chitosan microspheres (C-MNPs) are frequently used as carriers because of their significant biological (i.e., biodegradable, biocompatible, bioactive) and chemical properties (polycationic, hydrogel, contains reactive groups, such as -OH and -NH₂). Moreover, the hydrophilic properties of the C-MNPs play an important role in the preparation of oriented-immobilized cellulase based on the SRM system. The conventional immobilization of cellulase molecules on a single magnetic nanocarrier is simple, the chitosan was usually first coated on the magnetic nanocarriers for further combination with cellulase (Figure 1). Subsequently, the combined material based on Fe₃O₄ nanocarriers have received extensive attention in cellulase immobilization to improve enzyme activity, loading, and stability because of their low toxicity, biocompatibility, and easy synthesis (Jordan et al., 2011). Magnetite nanocarriers coated with silica and modified by organic-silanes, biocompatible, and with hydrophilic properties, are promising for cellulase immobilization.

The binding sites of enzymes on the surfaces of carriers depend on the chemical properties of the carriers. For non-covalent immobilization, the chemical structure of the skeleton and surface determines the applicability of carriers. The functional groups play a key role in the activity, stability, and selectivity of the enzyme, and the size, charge, polarity, and hydrophilicity/hydrophobicity of groups can affect their binding functions (Watanabe et al., 2010). Different properties of the ionic groups on the surfaces of carriers may result in different enzyme activities and further determine the structure of immobilized cellulase (Santos et al., 2015; Berlin et al., 2016; Frančič et al., 2016; Hui et al., 2016; Zhou et al., 2018). The chemistry properties of enzyme and carrier cause the oriented distribution of catalytic domain of enzyme from dispersion layer to diffusion layer during the immobilization process is shown in Figure 2. In this process, the CAGs directly participate in binding with enzyme molecules, but the carrier-bound inert groups (CIGs) are not directly involved. This interaction inevitably disturbs the maintenance of the natural conformation of the enzyme, leading to structural and functional changes in the enzyme molecules. No obvious stability change has been observed when the newly formed conformation is similar to that of the natural enzyme.

The covalent binding between carriers and catalytic cleft of the enzyme not only causes pore plugging of the surface, but also leads to the drag increment of in-diffusion. Although an initial high dosage of cellulase is added, the inhomogeneous distribution of the carrier surface structure results in the uncontrollable immobilization sites, and ineffective immobilization may lead to a significant loss of enzymatic activity and reduce the accessibility of the substrate to the functional sites. Moreover, the partition and mass transport limitations of nanocarriers may cause spatial variation in local reaction rates and further affect enzymatic hydrolysis (Du et al., 2017; Zeng et al., 2019). The chitosan molecules are mostly used because of the large number of -OH and amino groups (-NH₃), which are easier to co-precipitate with cellulase (Bindhu and Abraham, 2010; Urrutia et al., 2018; Saha et al., 2019; Mo et al., 2020). Moreover, surface modification is an important strategy for tuning the properties of nanocarriers. Surface modification can either alter the existing property or introduce new properties onto nanoparticles using various agents, such as organ siloxane, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and carbodiimide, as well as amino silanes, such as 3-aminopropyltriethoxysilane, aminoethyl aminopropyl polydimethylsiloxane, and silica (Chang et al., 2011; Gokhale et al., 2013; Riedel et al., 2017; Malar et al., 2018; Malar et al., 2020).

In most reports about hydrophobic ionic liquids, the enzymes are not dissolved but merely in a dispersed state and therefore regarded as a heterogeneous catalyst. Some hydrophilic ionic liquids can accelerate the dissolution of enzyme molecules, but cause the destruction of the protein secondary structure, leading to the inactivation of the enzyme (Fujita and Ohno, 2010; Moniruzzaman et al., 2010). In pure hydrophilic ionic liquids the enzymes can be dispersed at the monomolecular level. The hydrophilic proteins in almost anhydrous nonpolar solvents form suspensions, whereas proteins with extended hydrophobic surface segments form microemulsions in the same media, greatly reducing the catalytic efficiency of the enzyme (Zuev et al., 2003; Predvoditelev et al., 2010). Nonpolar hydrophobic solvents, such as heptane, octane, and benzene, do not cause the dehydration of biocatalysts. Therefore, the enzyme can maintain its catalytic activity (Muginova et al., 2010). Similarly, the catalytic activity of enzymes can be retained in the surfactant micelle system due to the water-oil amphiphilicity of surfactants (Muginova et al., 2010). Non-ionic surfactants can significantly accelerate the enzymatic hydrolysis of lignocellulose (Qing et al., 2010; Seo et al., 2011b; Eckard et al., 2012; Yiamsawas et al., 2017). For instance, Tween-20 can enhance the specific adsorption of cellulase, and the conversion efficiency of cellulose increased from 9 to 21% within 72 h when a high lignocellulosic substrate was added (Seo et al., 2011a). The prevention of non-productive enzyme adsorption onto lignin is the most widely investigated mechanism for this enhancement (Sipos et al., 2010; Lou et al., 2017). Recently, Djajadi et al. (2018) has proved that the adsorption of cellulases onto lignin substrates is reversible by nature, the reversible adsorption-desorption is existing in the process. But the non-productive adsorption caused by the ineffective combination will occupy large number of catalytic clefts of enzyme molecule which greatly hinder the enzymatic hydrolysis. Non-ionic surfactants can render lignin surfaces more hydrophilic by increasing their polar surface energy component, which can reduce the non-productive adsorption of cellulases onto lignocellulosic substrates caused by the ineffective combination between catalytic clefts of enzymes and lignin substrates (Jiang et al., 2017b), thereby promoting the enzymatic hydrolysis of lignocellulose. However, for the anionic surfactant-cellulase system, the adsorbed surfactants on the surface of cellulase cause a lower negative charge area, which further leads to negative catalytic activity due to the presence of sulfonic acid groups with a higher ionization degree (Yu and Zhang, 2016).

Furthermore, the effect of surfactants on cellulase hydrolysis is related to the concentration of surfactants (Zhou et al., 2015). In the enzymatic hydrolysis process, cellulose molecules are specifically adsorbed by the cellulose-binding domain (carbohydrate-binding module, CBM) and exert a driving force on the enzyme during the hydrolysis of cellulose (Liu et al., 2011; Tomme et al., 2015; Arslan et al., 2016). The adsorption of CBM can increase the cellulase concentration of the substrate surface by promoting the association of enzymes and substrates, but the non-covalent interactions (e.g., hydrogen bonds, electrostatic, and hydrophobic interactions) may lead to a nonproductive combination, because the random combination will occupy the active center of enzyme, resulting in the loss of catalytic activity. Ineffective adsorption can be reduced in the presence of surfactants due to the hydrophobic structure of surfactants, which can interact with the hydrophobic lignocellulosic substrates and form a coating (Kumar and Wyman, 2010; Li et al., 2012). However, contrasting results were obtained when different concentrations of surfactants were added to the enzymatic hydrolysis system. Some studies have suggested that a high concentration of surfactants can inhibit cellulase activity because strong hydrophobic interaction between the surfactant and cellulase can further reduce the effective adsorption of enzymes on cellulose (Wang Z. et al., 2013; Bao et al., 2019). However, the promotion effect of surfactant in enzymatic saccharification was observed in low concentration of lignosulfonate with low molecular weight and good sulfonation, which can be explained that the lignosulfonate can prevent the nonproductive binding of cellulase to lignin substrate, and the formed lignosulfonate-cellulase aggregate can also stabilize and enhance the binding of cellulase to lignin substrate (Lou et al., 2014).

The oriented immobilization of proteins on a solid support can effectively avoid its denaturation and keep its catalytic clefts fully exposed to solution, thus maximally preserving the bioaffinity or bioactivity. Liu and Yu (2016) has summarized the recent advances in oriented immobilization of proteins with a particular focus on antibodies and enzymes. However, the orientated immobilization of enzymes at the solvent interface is never involved. Thereby, the follow-up content will propose a novel method to achieve the oriented immobilization of cellulase in the SRM system.

Cellulase plays an important role in the production of fuel ethanol by the enzymatic hydrolysis of lignocellulose, and the immobilization of cellulase on the nanocarriers is an effective way to improve hydrolysis efficiency. However, the nanocarrier structure characteristics, solid-solid contact obstacles, external diffusion resistance, limited recycling frequency of nanocarriers, and nonproductive combination of enzyme active centers restricted the further improvement of hydrolysis efficiency in the complex multiphase system. Surfactants can promote the enzymatic hydrolysis of lignocellulose and play an important role in the preparation of nanocarriers. The special SRM system caused by the amphiphilicity in the oil-water reaction system can provide effective protection to obtain the immobilization of single-layer cellulase, which can effectively prevent the immobilization of cellulase and increase the effective attachment of immobilized cellulase and solid substrates.